Differentiation of adipose stromal cells into osteoblasts and treating bones

ABSTRACT

The invention provides methods and compositions for differentiating stromal cells from adipose tissue into cells having osteoblastic properties, and methods for improving a subject&#39;s bone structure. The methods comprise culturing stromal cells from adipose tissue in β-glycerophosphate and ascorbic acid and/or ascorbate-2-phosphate for a time sufficient to allow differentiation of said cells into osteoblasts. Such methods and compositions are useful in the production of osteoblasts for autologous transplantation into bone at a surgical site or injury. The compositions comprise adipose stromal cells, a medium capable of supporting the growth of fibroblasts and amounts of β-glycerophosphate and ascorbic acid and/or ascorbic-2 phosphate sufficient to induce the differentiation of said stromal cells into osteoblasts. 
     The invention further provides methods of identifying compounds that affect osteoblast differentiation. Such compounds are useful in the study of bone development and in the treatment of bone disorders, including bone fractures and osteoporosis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/061,214, filed Mar. 14, 2002, now allowed, which is a continuation ofU.S. application Ser. No. 09/554,868 filed May 19, 2000, now U.S. Pat.No. 6,391,297, which is a §371 national phase application based onInternational Patent Application No. PCT/US98/25449, filed Dec. 1, 1998,which claims benefit under 35 U.S.C. §119(e) to U.S. Provisional PatentApplication No. 60/067,334, filed Dec. 2, 1997.

FIELD OF THE INVENTION

This invention relates to methods and compositions for thedifferentiation of stromal cells from adipose tissue into osteoblasts,and uses thereof.

BACKGROUND OF THE INVENTION

Osteoporosis is responsible for about 1.5 million fractures each year inthe United States, of which about 300,000 are hip fractures. Fifty to75% of patients with hip fractures are unable to live independently,resulting in increased costs of care. Osteoporosis is characterized by agreater than normal loss of bone density as people age. This diseaseoccurs with a high frequency (>30% of females over age 60) in Westernand Asian cultures, and is increasing in prevalence as longevityincreases. While the exact cause of these bone repair disorders isunknown, it is clear the dynamic process of bone remodeling is disruptedin a process characterized by a decrease in osteoblastic (bone-producingcells) activity and an increase in osteoblastic (bone degrading cells)activity (Parfitt (1992) Triangle 31:99-110; Parfitt (1992) In Bone,volume 1, B. K. Hall, ed. Teleford Press and CRC Press, Boca Raton,Fla., p. 351-429).

The use of bone grafts is conventional practice in orthopedics,neurosurgery and dentistry, as well as in plastic/reconstruction surgeryand this utilization has been growing in frequency over the past twodecades. With the exception of blood, bone is the most frequentlytransplanted tissue with an estimated 500,000 bone grafts used in the USannually. Common orthopedic uses of bone grafts include the managementof non-unions and acute long bone fracture, joint reconstruction and tofacilitate fusion of vertebral motion segments in treating a variety ofspinal disorders (Lane (1987) Ortho Clin N Amer 18:213-225).

Currently, the most clinically acceptable grafting material isautologous bone. So-called autografts are often obtained from asecondary operative site. There are significant issues associated withautografts. These include lack of an adequate supply for large wounds ordefects. Elderly individuals with osteoporosis or osteopenia make theuse of an autograft problematic. The secondary morbidity associated withthe harvesting operation is high. These complications includeinfections, pelvic instability (the bone is often harvested from theiliac crest), hematoma, and pelvic fracture (Laurie et al. (1984) PlasRec Surg 73:933-938; Summers et al. (1989) J Bone Joint Surg71B:677-680; Younger et al. (1989) J Orthop Trauma. 3:192-195; Kurz etal. (1989) Spine 14:1324-1331). In addition, chronic pain at the donorsite is the second most frequently reported complication (Turner et al.(1992) JAMA 268:907-911). Finally, the ability to shape the autograft tothe defect/wound site is limited due to the rigid nature of thematerial.

Recent investigations have focused on the use of a variety of matrices,either inorganic such as hydroxyapatite (Flatley et al. (1983) ClinOrthop Rel Res 179:246-252; Shima et al. (1979) J Neurosurg 51:533-538;Whitehill et al. (1985) Spine 10:32-41; Herron, et al. (1989) Spine14:496-500; Cook et al. (1986) Spine 11:305-309; the contents of whichare incorporated herein by reference) or organic such as demineralizedbone matrix (DBM) (reviewed in Ashay et al. (1995) Am J Orthop24:752-761; the contents of which are incorporated herein by reference).These matrices are thought to be osteoconductive (facilitate theinvasion of bone forming cells in an inert matrix) or osteoinductive(induce the transformation of recruited precursor cells to osteoblasts).A number of successful clinical outcomes have been observed with some ofthese products approved for use clinically by the Food and DrugAdministration. In spite of these successes, a number of issues remainfor the utility of these matrices. The first is the variable subjectresponse to DBM. Also these matrices take much longer than autologousbone transplantation to develop significant structural integrity andbear load effectively.

An alternative to transplantation and the use of simple matrices is theadmixture of bone marrow or bone marrow stromal cells with DBM. Ideallythe cells and DBM will be derived from the same subject althoughallogeneic DBM has already been used clinically with initial success(Mulliken et al. (1981) Ann Surg 194:366-372; Kaban et al. (1982) J OralMaxillofac Surg 40:623-626). Transplantation methods using autologousbone marrow cells with allogeneic DBM have yielded good results(Connolly (1995) Clin Orthop 313:8-18). However, issues that may impactthe widespread use of these techniques include potential forcontamination by non-self materials, the acceptability of the patientfor donating bone marrow, and the potential complications that arisefrom bone marrow aspirations and depletion of bone marrow from thesource.

A number of groups have shown that bone marrow stromal cells and celllines derived thereof are capable of differentiating into cellsbiochemically and morphologically similar to osteoblasts (Dorheim et al.(1993) J Cell Physiol 154:317-328; Grigoriadis et al. (1988) J Cell Biol106:2139-2151; Benayahu et al. (1991) Calcif Tiss Int. 49:202-207; thecontents of which are incorporated by reference). In most cases,fibroblast-like cells were isolated from human or animal bone marrow andplated onto standard tissue cultureware. Generally, a standard mediaformulation, such as Dulbecco's Modified Eagle's Medium (DMEM) plusfetal calf serum 10-20% and antibiotics is used to select for theenrichment of these cells (Ashton et al. (1980) Clin Orthop 151:294-307;Sonis et al. (1983) J Oral Med 3:117-120). Cells were then stimulated todifferentiate into osteoblasts by changing the medium to one containing5-20% fetal calf serum, 2-20 mM β-glycerophosphate and 20-75 μM ascorbicacid or ascorbic-2-phosphate (Asahina et al. (1996) Exp Cell Res222:38-47; Yamaguchi et al. (1991) Calcif Tissue Int 49:221-225; thecontents of which are incorporated herein by reference). After 14-21days in culture, many of these cell types and cell lines willmineralized matrices on the cultureware as evidenced by positive vonKossa staining. Other phenotypic indicators of osteoblast lineageinclude elevated secreted alkaline phosphatase activity; the presence ofsecreted osteocalcin in the media; and the increased expression-ofseveral genes thought to be specifically expressed in osteoblasts,including osteocalcin, osteopontin, and bone sialoprotein (Stein et al.(1990) FASEB J 4:3111-3123; Dorheim et al. (1993) J Cell Physiol154:317-328; Asahina et al. (1996) Exp Cell Res 222:38-47; Yamaguchi etal. (1991) Calcif Tissue Int 49:221-225).

There have been a number of detailed studies carried out in severallaboratories demonstrating that transplanted bone marrow stromal cellscan form ectopic bone (Gundle et al. (1995) Bone 16:597-603; Haynesworthet al. (1992) Bone 13:81-89; Boynton et al. (1996) Bone 18:321-329). Forexample, human and murine bone marrow stromal fibroblasts have beentransplanted into immunodeficient SCID mice (Krebsbach et al. (1997)Transplantation 63:1059-1069; Kuznetsov et al. (1997) J Bone Min Res12:1335-1347). Using antibody and histochemical markers, it wasdemonstrated that the donor bone marrow stromal cells account for thenewly developed osteoblasts at sites of ectopic bone formation in thepresence of an inductive matrix. Murine cells formed bone in thepresence of hydroxyapatite/tricalcium phosphate particles (HAITCP),gelatin, poly-L-lysine, and collagen. In contrast, human stromal cellsefficiently formed bone only in the presence HA/TCP. No exogenous BMPwas required in these studies.

Bone formation is not limited to the skeleton. For example, theintroduction of ceramic or demineralized bone matrix into intramuscular,subrenal capsular or subcutaneous sites will result in bone formation ifthe area is simultaneously expressing bone morphogenetic protein (Urist(1965) Science 150:893.-899). These results suggest that cells presentin these tissues have some capability of forming bone precursor cellsunder the proper environmental conditions.

Ectopic bone formation in soft tissue such as fat is a rare pathologiccondition observed in patients with fibrosis ossificans progressiva, aninherited disease. While the etiology of the disease is not completelyunderstood, it arises in part from the abnormal expression of BMP bylymphocytes localized to sites of soft tissue injury (Kaplan et al.(1997) J Bone Min Res 12:855; Shafritz et al. (1996) N Engl J Med335:555-561]). Bone formation is also observed on rare occasions inlipomas (Katzer (1989) Path Res Pract 184:437-443).

The stromal-vascular fraction isolated from adipose tissue aftercollagenase treatment has been demonstrated to contain a large quantityof preadipocytes, or cells that are predisposed to differentiate intoadipocytes (Hauner et al. (1989) J Clin Invest 34:1663-1670). Thesecells can spontaneously differentiate into adipocyies at relatively lowfrequency or respond to adipogenic agonists such as thiazolidinedionesto a much higher frequency of differentiation (Halvorsen (1997)Strategies 11:58-60; Digby (1997) Diabetes 4:138-141). There is evidenceto suggest that stromal cells exhibit a reciprocal pattern ofdifferentiation between these lineages (Gimble et al. (1996) Bone19:421-428; Bennett et al. (1991) J Cell Sci 99:131-139; Beresford etal. (1992) J Cell Sci 102:341-351). Specifically, adipogenesis isaccompanied by reduced osteoblastic potential while osteogenesis isaccompanied by reduced adipogenic potential.

Under certain conditions, bone marrow stromal cells can bedifferentiated into adipocytes. In fact, several bone stromal cell lineshave been extensively characterized with respect to this ability (Gimbleet al. (1990) Eur J Immunol 20:379-387; Gimble et al. (1992) J CellBiochem 50:73-82; Gimble et al. (1996) Bone 19:421-428). However, priorto the instant invention, it was not known that stromal cells isolatedfrom adipose tissue could be made to differentiate into osteoblasts.

SUMMARY OF THE INVENTION

The invention provides methods and compositions for differentiatingstromal cells from adipose tissue into cell having osteoblasticproperties, and methods for improving a subject's bone structure. Themethods comprise culturing stromal cells from adipose tissue inβ-glycerophosphate and ascorbic acid and/or ascorbate-2-phosphate for atime sufficient to allow differentiation of said cells into osteoblasts.Such methods and compositions are useful in the production ofosteoblasts for autologous transplantation into bone at a surgical siteor injury. The compositions comprise adipose stromal cells, a mediumcapable of supporting the growth of fibroblasts and differentiationinducing amounts of β-glycerophosphate and ascorbic acid and/orascorbic-2 phosphate.

The invention further provides methods of identifying compounds thataffect osteoblast differentiation. Such compounds are useful in thestudy of bone development and in the treatment of bone disorders,including bone fractures and osteoporosis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the morphological changes that occur in human adiposestromal cells induced to differentiate into osteoblasts by treatmentwith osteoblast differentiation medium.

FIG. 2 shows adipose stromal cells that have been treated withosteoblast differentiation medium or adipocyte differentiation mediumand stained with Oil Red O (FIGS. 2A and 2C) or by the von Kossa method(FIGS. 2B and 2C).

FIG. 3 shows the time course of concentrations of osteocalcin and leptinsecreted into the media upon treatment of adipose stromal cells withstromal cell medium (PA), adipocyte medium (AD) or osteoblast medium(OST).

FIG. 4 shows the concentrations of alkaline phosphatase secreted byadipose stromal cells cultured for twenty-one days in stromal cellmedium (SC), adipocyte differentiation medium (AD) or osteoblastdifferentiation medium (OST).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for differentiating adiposestromal cells into osteoblasts. The osteoblasts produced by the methodsof the invention are useful in providing a source osteoblasts forresearch or transplantation into a subject's bone, at the site ofsurgery or fracture. Thus in one aspect, the invention provides a methodof differentiating adipose stromal cells into osteoblasts, comprising:culturing said cells in a composition which comprises a medium capableof supporting the growth of fibroblasts and differentiation inducingamounts of β-glycerophosphate and ascorbic acid and/or ascorbic-2phosphate.

In another aspect, the invention provides compositions for thedifferentiation of adipose stromal cells into osteoblasts. Suchcompositions comprise: adipose stromal cells, a medium capable ofsupporting the growth of fibroblasts and amounts of β-glycerophosphateand ascorbic acid and/or ascorbic-2 phosphate sufficient to induce thedifferentiation of said stromal cells into osteoblasts.

“Adipose stromal cells” refers to stromal cells that originate fromadipose tissue. By “adipose” is meant any fat tissue. The adipose tissuemay be brown or white adipose tissue. Preferably, the adipose issubcutaneous white adipose tissue. Such cells may comprise a primarycell culture or an immortalized cell line. The adipose tissue may befrom any organism having fat tissue. Preferably the adipose tissue ismammalian, most preferably the adipose tissue is human. A convenientsource of human adipose tissue is from liposuction surgery, however, thesource of adipose tissue or the method of isolation of adipose tissue isnot critical to the invention. If osteoblasts are desired for autologoustransplantation into a subject, the adipose tissue will be isolated fromthat subject. “Differentiation inducing amounts of β-glycerophosphateand ascorbic acid and/or ascorbic-2 phosphate” refers to concentrationsof β-glycerophosphate and (ascorbic acid and/or ascorbic-2 phosphate),that when supplied in a medium capable of supporting the growth offibroblasts (e.g., NIH-3T3 cells, human adipose stromal cells and thelike), will induce the differentiation of said stromal cells intoosteoblasts over a time period of about five days to eight weeks.Optimal concentrations and lengths of treatment may be determined by thepractitioner through the use of known assays for differentiatedosteoblasts. Such assays include, but are not limited to those thatassess morphological or biochemical characteristics (e.g., secretedosteocalcin or other osteoblast-specific proteins or RNA).

The concentration of ascorbic acid and/or ascorbic-2-phosphate refers toany combined concentration of these compounds that total the statedconcentration. For example, the definition of “50 μM ascorbic acidand/or ascorbic-2 phosphate” includes, but is not limited to, suchpermutations as: 50 μM ascorbic acid; 50 μM ascorbic-2 phosphate; 10 μMascorbic acid and 40 μM ascorbic-2 phosphate; or 40 μM ascorbic acid and10 μM ascorbic-2 phosphate.

Preferably the medium contains about 2-20 mM β-glycerophosphate andabout 20-75 μM ascorbic acid and/or ascorbic-2 phosphate. Morepreferably, the medium contains about 5-15 mM β-glycerophosphate andabout 40-60 mM ascorbic acid and/or ascorbic-2 phosphate. Mostpreferably, the medium contains about 10 mM β-glycerophosphate and about50 μM ascorbic acid and/or ascorbic-2 phosphate.

Any medium capable a supporting fibroblasts in cell culture may be used.Media formulations that will support the growth of fibroblasts include,but are not limited to, Dulbecco's Modified Eagle's Medium (DMEM), alphamodified Minimum Essential Medium (αMEM), and Basal Medium Essential(BME) and the like. Typically, 5-20% Fetal Calf Serum (FCS) will beadded to the above media in order to support the growth of fibroblasts.However, a defined medium could be used if the factors in FCS necessaryfor fibroblast growth were identified and provided in the growth medium.

Media useful in the methods of the invention may contain one or morecompounds of interest, including, but not limited to, antibiotics,compounds that are osteoinductive, osteoconductive, or promote growth ordifferentiation, such as bone morphogenetic proteins or other growthfactors. Examples of bone morphogenetic proteins include, but are notlimited to, osteogenic protein-1, BMP-5, osteogenic, osteoinductivefactor and bone morphogenetic protein-4 (Asahina et al. (1996) Exp CellRes 222:38-47; Takuwa (1991) Biochem Biophys Res Com 174:96-101; Chen(1991) J Bone Min Res 6:1387-1390; Sampath (1992) J Biol Chem267:20352-20362; Wozney et al. 1988 Science 242:1528-1534, the contentsof which are incorporated herein by reference), and the like.

Preferably, the adipose tissue is treated so that the stromal cells aredissociated from each other and from other cell types, and precipitatedblood components are removed. Typically, dissociation into single viablecells may be achieved by treating adipose tissue with proteolyticenzymes, such as collagenase and/or trypsin, and with agents thatchelate Ca²⁺. Stromal cells may then be partially or completely purifiedby a variety of means known to those skilled in the art, such asdifferential centrifugation, fluorescence-activated cell sorting,affinity chromatography, and the like. The partially or completelyisolated stromal cells may then cultured in a media that will supportthe growth of fibroblasts for a period of between eight hours to up tofive cell passages prior to treatment with media containingβ-glycerophosphate and ascorbic acid and/or ascorbic-2-phosphate.

The stromal cells will be cultured in media containingβ-glycerophosphate and ascorbic acid and/or ascorbic-2-phosphate for atime sufficient to induce differentiation into osteoblasts. The lengthof treatment with β-glycerophosphate and ascorbic acid and/orascorbic-2-phosphate required for differentiation of stromal cells intoosteoblasts is dependent upon a number of factors. Such factors include,but are not limited to, the concentrations of β-glycerophosphate andascorbic acid and/or ascorbic-2-phosphate used, the medium used, thesource of adipose tissue or stromal cells, the initial density ofplating, the presence or absence of growth factors or bone morphogeneticproteins and the like. The concentration of β-glycerophosphate andascorbic acid or ascorbic-2-phosphate and other conditions and factorsmay be optimized by the practitioner. Optimal concentrations andtreatment times may be determined by measuring the percentage of cellsthat have differentiated into osteoblasts. This percentage may bemonitored by morphological and biochemical assays and indices known tothose skilled in the art. Such assays and indices include, but are notlimited to, those that assess morphological or biochemicalcharacteristics, such as the presence of calcium deposits orosteoblast-specific proteins or RNAs, von Kossa staining, osteocalcinsecretion and alkaline phosphatase secretion.

Osteoblasts derived from adipose tissue stromal cells may be introducedinto the bone of a human or animal subject at the site of surgery orfracture. Introduction of osteoblasts to bone is useful in the treatmentof bone fractures and bone disorders, including osteoporosis. Thus, inanother aspect, the invention is directed to a method of improving asubject's bone structure, comprising:

-   -   a) culturing stromal cell from adipose tissue in a composition        which comprises a medium capable of supporting the growth of        fibroblasts and differentiation inducing amounts of        β-glycerophosphate and ascorbic acid and/or ascorbic-2        phosphate; and    -   b) introducing said osteoblasts into a surgery or fracture site        of said subject.

Preferably, the stromal cells are isolated from the adipose tissue ofthe subject into which the differentiated osteoblasts are to beintroduced. However, the stromal cells may also be isolated from anorganism of the same or different species as the subject. The subjectmay be any organism having bone tissue. Preferably the subject ismammalian, most preferably the subject is human.

The stromal cells or osteoblasts may be stably or transientlytransformed with a nucleic acid of interest prior to introduction into asurgery or fracture site of the subject. Nucleic acid sequences ofinterest include, but are not limited to those encoding gene productsthat enhance the growth, differentiation and/or mineralization ofosteoblasts. For example, an expression system for bone morphogeneticprotein 4, can be introduced into the preadipocytes in a stable ortransient fashion for the purpose of treating non-healing fractures orosteoporosis. Methods of transformation of stromal cells and osteoblastsare known to those skilled in the art, as are methods for introducingosteoblasts into a bone at the site of surgery or fracture.

The osteoblasts may be introduced alone or in admixture with acomposition useful in the repair of bone wounds and defects. Suchcompositions include, but are not limited to bone morphogeneticproteins, hydroxyapatite/tricalcium phosphate particles (HA/TCP),gelatin, poly-L-lysine, and collagen. For example, osteoblastsdifferentiated from adipose stromal cells may be combined with DBM orother matrices to make the composite osteogenic (bone forming in it ownright) as well as osteoinductive. Similar methods using autologous bonemarrow cells with allogeneic DBM have yielded good results (Connolly(1995) Clin Orthop 313:8-18).

A further object of the invention is to provide methods for theidentification and study of compounds that enhance the differentiationof stromal cells into osteoblasts. Compounds that enhance thedifferentiation of osteoblasts may play a role in the treatment ofvarious bone disorders, including fractures and osteoporosis. Inaddition, compounds found to induce osteoblast differentiation areuseful in for differentiating cells in vitro or in vivo. Conversely,compounds or factors found to block osteoblast differentiation may beuseful in certain disease states where idiosyncratic bone production,such as Paget's disease or chondroosteoblastic metaplasia, may betreated by such compounds. Thus, in another aspect, the invention isdirected to a method of identifying compounds that affect osteoblastdifferentiation, comprising:

-   -   a) culturing adipose stromal cells in the presence and absence        of a compound to be tested for effect on osteoblast        differentiation in a composition which comprises a medium        capable of supporting the growth of fibroblasts and        differentiation inducing amounts of β-glycerophosphate and        ascorbic acid and/or ascorbic-2 phosphate; and    -   b) comparing osteoblast differentiation in said cells cultured        in the presence of said compound to that of said cells cultured        in the absence of said compound.

Any compound may be tested for its ability to affect the differentiationof stromal cells into osteoblasts. Appropriate vehicles compatible withthe compound to be tested are known to those skilled in the art and maybe found in the current edition of Remington's Pharmaceutical Sciences,the contents of which are incorporated herein by reference.

The results of the tests can be compared with results using knowndifferentiation promoting agents, such as osteogenic protein 1 and bonemorphogenetic protein-4 (Asahina et al. (1996) Exp Cell Res 222:38-47;Takawa (1991) supra; Chen (1991) supra; Sampath (1992) supra; Wozney etal. (1988) Science 242:1528-1534), which are known to promotedifferentiation of osteoblasts by increasing the expression ofosteoblast markers such as osteocalcin, a definitive marker ofosteoblast function (Celeste (1986) Proc Natl Acad Sci 87:9843-9872;Stein et al. (1990) FASEB J 4:3111-3123). Also, the results of suchtests may be compared to known osteoblast differentiation inhibitorssuch as TNF-alpha, which results in complete or partial blocking of theconversion of stromal cells into osteoblasts.

The features and advantages of present invention will be more clearlyunderstood by reference to the following examples, which are not to beconstrued as limiting the invention.

EXAMPLES Example I Isolation of Stromal from Human Adipose Tissue

Human stromal cells were isolated from adipose tissue according to theprocedures described by Rodbell (1964) J Biol Chem 239:375 and Hauner etal. (1989) J Clin Invest 84:1663-1670. Briefly, human adipose tissuefrom subcutaneous depots was removed by liposuction surgery. The adiposetissue was then transferred from the liposuction cup into a 500 mlsterile beaker and allowed to settle for about 10 minutes. Precipitatedblood was removed by suction. A 125 ml volume (or less) of the tissuewas transferred to a 250 ml centrifuge tube, and the tube was thenfilled with Krebs-Ringer Buffer. The tissue and buffers were allowed tosettle for about three minutes or until a clear separation was achieved,and then the buffer was removed by aspiration. The tissue was washedwith Krebs-Ringer Buffer an additional four to five times or until itwas orange-yellow in color and the buffer was light tan in color.

The cells of the adipose tissue were dissociated by collagenasetreatment. Briefly, the buffer was removed from the tissue and replacedwith a 2 mg collagenase/ml Krebs Biffer (Worthington, Mass., USA, typeI) solution at a ratio of 1 ml collagenase solution/ml tissue. The tubeswere incubated in a 37° C. water bath with intermittent shaking for 30to 35 minutes.

Stromal cells were isolated from other components of the adipose tissueby centrifugation for 5 minutes at 500×g at room temperature. The oiland adipocyte layer was removed by aspiration. The remainingstromal-vascular fraction was resuspended in approximately 100 ml ofphosphate buffered saline (PBS) by vigorous swirling, divided into 50 mltubes and centrifuged for five minutes at 500×g. The buffer wascarefully removed by aspiration, leaving the stromal cells. The stromalcells were then resuspended in stromal cell medium (DMEM (Morton (1970)In Vitro 6:89-108; Dulbecco (1959) Virology 8:396)/Ham's F-10 medium(Ham (1963) Exp Cell Res 29:515) (1:1, v/v); 10% (v/v) fetal calf serum;15 mM HEPES, pH 7.4; 60 U/ml penicillin; 60 U/ml streptomycin; 15 μg/mlamphotericin B), plated at an appropriate cell density and incubated at37° C. in 5% CO₂ overnight. Once attached to the tissue culture dish orflask, the cultured stromal cells may be used immediately or maintainedin culture for up to 5 passages before being induced to differentiateinto osteoblasts as described in Example 2 below.

Example 2 Differentiation of Extramedullary Adipose Stromal Cells IntoOsteoblasts

Adipose stromal cells were isolated as described in Example 1 and thentreated as follows to induce differentiation into osteoblasts. Stromalcells were plated in 24-well and/or 6-well tissue culture plates instromal cell medium (see above) at a density of about 22,000 cells/cm².After 24 hours, the stromal cell medium was replaced with osteoblastdifferentiation medium (DMEM with 10% fetal bovine serum (v/v); 10 mMβ-glycerophosphate; 50 μg/ml ascorbate-2-phosphate; 60 U/ml penicillin;60 U/ml streptomycin; 15 μg/ml amphotericin B). The osteoblastdifferentiation medium was replaced with fresh medium every 3 days for 3weeks. When changing the media, one ml of conditioned media wascollected and stored at −80° C. for later analysis of secreted factors.Alternatively, stromal cells isolated from adipose tissue were inducedto differentiate into adipocytes according to the method of Hauner etal. (1989 J Clin Invest 34:1663-1670) by treatment with adipocytedifferentiation medium.

Microscopic examination of cells treated with osteoblast medium asdescribed above revealed morphological changes consistent with theappearance of osteoblasts (FIG. 1). After prolonged culture (21-28days), several multi-cellular nodules formed in each culture well. Thegrainy appearance of the osteoblast culture, indicates the presence ofcalcium phosphate deposits and pre-bone structures. Because stromalcells isolated from adipose tissue also have the potential todifferentiate into adipocytes when treated with an adipocytedifferentiation medium, the cells treated with osteoblast medium werealso examined for the presence of adipocytes. No obvious adipocytes inthe cultures treated with osteoblast medium, as indicated by the lack ofoil droplets appearing in the cytoplasm and lack of cells having thecharacteristic rounded adipocyte morphology.

Cells treated with the osteoblast differentiation medium were stained bythe von Kossa method to determine whether the stromal cells haddifferentiated into osteoblasts. Briefly, fetal calf serum was seriallydiluted out of the medium by exchanging 80% of the medium several timeswith serum-free medium. The cells were fixed in 5% formaldehyde and thenwashed several times with PBS to remove any remaining serum. The fixedcells were incubated in 100% ethanol at 4° C. for about 10 minutes. Theethanol was then removed and the fixed cells were incubated in 0.5 ml 5%silver nitrate for 10 minutes under UV light at 254 nm. The cells werethen rinsed 2-3 times in distilled water, incubated in 5% sodiumthiosulfate for 5 minutes and then rinsed with water. The stained cellscan be stored in 50% glycerol indefinitely. The results of von Kossastaining are shown in FIGS. 2B and 2C. Only cells receiving osteoblastmedium stained positive and turned dark.

Oil Red O staining was performed as follows. Plates were rinsed withphosphate buffered saline several times to remove serum or bovine serumalbumin in the culture medium. The cells were then fixed in methanol or10% formaldehyde in phosphate buffered saline for 15 minutes to 24hours. An Oil Red O working solution was prepared by adding 6 ml of astock solution (0.5 g Oil Red O in 100 ml isopropanol) to 4 ml of dH₂O.The working solution was kept for an hour at room temperature beforefiltering through a Whatman #1 filter. Cells were stained withapproximately 3 ml/100 mm plate or 1 ml/well in 6-well plate for 1hour-at room temperature and then rinsed several times with H₂O. All ofthe remaining wash water was removed. 150 μl/well isopropanol was addedand the plate was incubated at room temperature for 10 minutes. Theisopropanol was pipetted up and down several times, to ensure that allof the oil red O was in solution. The optical density was measured at500 nM.

Cells receiving adipocyte differentiation medium stained with oil red Oas described below displayed the characteristic red color indicatingaccumulation of lipid (FIG. 2A). Preadipocytes showed very small oildroplets accumulating after 4 weeks in culture. Cells receivingosteoblast medium showed some non-specific background oil red O,however, the staining was not associated with cells (FIG. 2C). Theseresults suggest that stromal cells differentiating into osteoblasts donot have detectable adipocyte morphology. Lack of such neutral lipidaccumulation is an indicator of loss of adipocyte function and lineage.

Biochemical changes indicative of osteoblast differentiation were alsoassessed. Osteocalcin is an osteoblast-specific secreted protein thatcan be measured by ELISA (Intact Human Osteocalcin K EIA kit, catalognumber BT-460, Biomedical Technologies, Inc., Stoughton, Mass.). Mediaobtained from osteoblasts, preadipocytes, and adipocytes were examinedfor the presence of secreted osteocalcin. Briefly, conditioned medium (2ml of medium from 40,000 cells after 72 hours) was collected and 20 μlof each medium was used for the assay. As shown in FIG. 3, there isincreasing osteocalcin found in the media of the osteoblasts, whilelittle or no osteocalcin secretion is seen in the adipose stromal cellsand adipocytes.

Secreted leptin peptide was measured by a commercially available ELISAkit. Conditioned medium (2 ml of medium from 40,000 cells wasconditioned for 72 hours) was collected and 100 μl of each medium wasused for the assay following protocol suggested by the manufacturer. Asexpected, leptin is secreted into the media is increased duringadipocyte differentiation but not in during osteoblast differentiation.The presence of leptin, an adipocyte-specific secreted protein, in theconditioned media is a clear marker for adipocyte activity. Osteoblastsand stromal cells fail to secrete any detectable leptin into the media,indicating a lack of adipocyte lineage, while only cells undergoingadipocyte differentiation secrete leptin after 2 weeks.

Another indicator of osteoblast lineage is the ability to secretealkaline phosphatase. Cells were prepared and incubated as describedabove and the conditioned media assayed for alkaline phosphataseactivity using a commercially available alkaline phosphatase assay kit(Sigma Diagnostic Inc., catalog number 104-LS, St Louis, Mo.). As shownin FIG. 4, the preadipocytes had a basal level of secreted alkalinephosphatase activity. Upon differentiation, the adipocytes lost thisbasal level of secreted activity while the osteoblasts showed anincrease in the levels of alkaline phosphatase.

Example 3 Identification of Compounds Affecting OsteoblastDifferentiation

Cultures of stromal cells isolated from adipose tissue according to themethod described in Example 1 were used to study the effect compounds ofinterest on osteoblast differentiation. Osteoblasts were differentiatedfrom adipose stromal cells as in Example 2, but in the presence andabsence of a compound of interest. Differentiation was measured byassays for secreted osteocalcin. Compounds found to positively affectosteoblast differentiation using the method of this example include bonemorphogenetic protein 4 and osteopontin-I (data not shown). Theseresults are in agreement with the previous findings of Wozney et al.1988 Science 242:1528-1534;.Asahina et al. 1996 Exp Cell Res 222:38-47;and Cook et al. 1996 Clin Orthop. 324:29-38. Compounds that enhanceosteoblast differentiation may be used to enhance the bone structure ofa subject, as discussed below.

Example 4 Use of Osteoblasts in Bone Repair

Stromal cells are isolated from adipose tissue using liposuction frompatients suffering from non-healing fractures or osteoporotic fracturesusing the method of Example 1. The preadipocytes are induced todifferentiate to osteoblasts in vitro using the method described inExample 2. After 7-21 days, the differentiated osteoblasts areharvested, for example, by trypsin treatment or mechanical scraping ofthe differentiated cells from a tissue culture plate and then areconcentrated by centrifugation at 3000×g for 10 min at 4-20° C. understerile conditions. The harvested cells are resuspended in a collagen orMatrigel™ solution and are then injected directly into the fracture orsurgery site using a 20 gauge or larger bore needle. Alternatively,before injection, the cells are mixed with DBM or a ceramic matrix suchas ProOsteon 2000™ (Interpore Cross, Irvine, Calif.) or Collagraft™(Zimmer Inc., Warsaw, Ind.). The amount of cells used will depend uponthe surface area of the fracture and the nature of the fracture.Multiple treatments may be necessary depending upon the speed ofrecovery of the fracture desired. The result is decreased time tohealing and increased bone density around the fracture site.

Example 5 Use of Genetically Altered Osteoblasts for Fractures orOsteoporosis

Stromal cells are isolated as described in Example 1 and geneticmaterial (e.g., DNA encoding useful gene products, such as bonemorphogenetic proteins, operably linked to a promoter) is introducedinto the stromal cells using standard transfection methods such ascalcium chloride (Maniatis et al. (1982) Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.). Such aprotocol has been developed using Effectene reagent and is describedherein. In a microcentrifuge tube,0.1-2.0 ug pCMV-βgal (Stratagene, Inc.La Jolla, Calif.) is added to 150 μl of Buffer EC (Qiagen Effectene™kit; catalog number 301425, Qiagen Corp.). This allows the DNA tocondense. 8 μl enhancer (Qiagen Effectene kit) is added to the condensedDNA. The tube containing the condensed DNA is then vortexed for onesecond and allowed to incubate at room temperature for 2-5 minutes. Thetube is centrifuged briefly to remove drops at the top of the tube. 10μl Effectene™ is added, the tube is vortexed for 10 seconds, andincubated at room temperature for 5-10 minutes. Following a 5-10 minuteincubation time, 1 ml of medium is added to the DNA mixture.

120 μl of the old medium is removed from the cells and 70 μl of freshmedium is added. 25 μl of the DNA mixture is then to each well. Thecells are incubated at 37° C. for about 5 hours. However, Effectene isnot toxic and may be left on the cells for any period of time.

The cells are then rinsed once with 80 μl fresh medium and assayed at 72hours post-infection for β-galactosidase activity using the methoddescribed by Maniatis et al. (1982). Such cells may be differentiatedinto osteoblasts under the methods described in Example I. Alternativelyor the DNA maybe introduced directly into cells differentiated intoosteoblasts.

Other methods of introducing nucleic acid sequences into cells may alsobe used. For example, adenovirus, may be used to introduce DNA into thestromal cells similarly to the protocols described by Becker et al.(1994) Meth Cell Biol 43:161-189 and Meunier-Durmont et al. (1996) EurBiochem 237:660-667. The cells are then treated so they differentiateinto osteoblasts as described above in Example I. Alternatively, thedifferentiated osteoblasts will be amenable to infection by viralparticles. An addition of an antibiotic selection marker allowsenrichment for cells bearing the introduced genetic material. Thederived osteoblasts bearing the introduced genetic material are thenintroduced to fracture and osteoporotic bone marrow as described abovein Example III.

All publications mentioned in the specification are indicative of thelevel of those skilled in the art to which this invention pertains. Allpublications are herein incorporated by reference to the same extent asif each individual publication was specifically and individuallyindicated to be incorporated by reference.

While the invention has been described with reference specificembodiments, it will be appreciated that numerous variations,modifications, and embodiments are possible, and accordingly, all suchvariations, modifications, and embodiments are to be regarded as beingwithin the spirit and scope of the invention.

The invention claimed is:
 1. A method of treating a bone wound, bonefracture or bone disorder in a subject in need of increased boneformation, comprising: a) culturing adipose stromal cells (ASCs) in acell culture medium capable of supporting the growth of fibroblasts, themedium comprising an effective amount of (β-glycerophosphate andascorbic acid and/or ascorbic-2 phosphate to induce differentiation ofthe ASCs, wherein the differentiated ASCs have positive von Kossastaining, secrete osteocalcin and/or have an elevated secreted alkalinephosphatase activity, and b) introducing the differentiated adiposestromal cells into a site of a bone wound, bone fracture or bonedisorder of the subject in need of increased bone formation, therebytreating the wound, fracture or disorder.
 2. The method of claim 1,wherein said amounts are about 2-20 mM (β-glycerophosphate and about20-75 μM ascorbic acid and/or ascorbic-2 phosphate.
 3. The method ofclaim 2, wherein said amounts are about 5-15 mM (β-glycerophosphate andabout 40-60 μM ascorbic acid and/or ascorbic-2 phosphate.
 4. The methodof claim 3, wherein said amounts are about 10 mM β-glycerophosphate andabout 50 μM ascorbic acid and/or ascorbic-2 phosphate.
 5. The method ofclaim 1, wherein said adipose stromal cells are isolated from saidsubject.
 6. The method of claim 1, wherein said medium is selected fromthe group consisting of: DMEM, αMEM and BME.
 7. The method of claim 1,wherein said medium further comprises about 5-20% fetal calf serum. 8.The method of claim 1, wherein said subject is mammalian.
 9. The methodof claim 8, wherein said subject is human.
 10. The method of claim 1,wherein said differentiated stromal cells are osteoblasts and areintroduced in admixture with a composition useful in the repair of bonewounds, bone defects and/or bone disorders.